Radical Rewrite (Matthieu Landon in Church Lab)

Researchers in the laboratory of George Church, Robert Winthrop Professor of Genetics at Harvard Medical School and a core faculty member at the Wyss Institute for Biologically Inspired Engineering at Harvard, are working to produce the most modified bacterial genome to date. The researchers believe the method they developed will help others who are trying to make many edits at once to any organism’s genome.

Harvard Medicine News spoke with Church lab postdoctoral researcher Nili Ostrov and graduate student Matthieu Landon about how the research is being done and what the achievement will mean for the field of genome engineering.

LANDON: Previously, our lab used precision genome editing technologies to replace about 300 instances of one particular codon each time it appeared throughout the genome of an E. coli bacterium. Codons are three-letter DNA sequences that code for amino acids, the building blocks of proteins. Replacing that single codon was a huge undertaking.

Here, we wanted to make over 60,000 genome changes so we could replace not just one but seven codons at once. Rather than gradually editing an existing genome, we did this by computationally designing and synthesizing an entirely new E. coli genome, with all the necessary genetic modifications already built in.

HMS: Did it work?

OSTROV: Though we are not done yet, so far it works very well. We constructed the synthetic genome in 87 overlapping segments and we are testing them one by one in living cells. Our goal is to confirm that, when we insert the recoded genes into a cell, the cell stays alive and expresses proteins normally. Then we will combine all the segments into a single fully-recoded genome.

LANDON: We’ve scrutinized about two-thirds of the genome so far (55 out of the 87 segments), and to our great happiness and surprise, we have found problems in only 13 of 2,229 genes tested. That is a very small number considering the extent of changes we made, which gives us confidence that our method is working and that we can complete the final organism.

HMS: Why not just test the whole genome at once?

OSTROV: If we had made all of the changes at the same time, and then the genome didn’t work, it would have been very difficult to pinpoint exactly which of the 4,000 altered genes was troublesome. On the other hand, testing individual genes would have taken a very long time.

LANDON: This is why we’ve chosen an intermediate segment size for testing. Working with these larger pieces of DNA allows us to test batches of genes at the same time—but not too many—so that we can quickly troubleshoot any problems. This was a critical aspect of our assembly method.